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Atypical Protein Kinase C‐specific Activity Reporter Reveals Novel Activation Mechanism of Atypical Protein Kinase C by Sphingosine 1‐phosphate
Author(s) -
Kajimoto Taketoshi,
Caliman Alisha D.,
Tobias Irene S.,
Okada Taro,
McCammon J. Andrew,
Nakamura Shunichi,
Newton Alexandra C.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.662.1
Subject(s) - isozyme , protein kinase c , diacylglycerol kinase , sphingosine , microbiology and biotechnology , biochemistry , biology , chemistry , kinase , enzyme , receptor
The protein kinase C (PKC) family comprises 9 members; conventional PKC isozymes (α, β γ), novel PKC isozymes (δ, ɛ, θ, η), and atypical PKC isozymes (ζ, ι/λ). It is well established that conventional and novel PKC isozymes are activated by second messengers, diacylglycerol and, for the conventional PKC isozymes, calcium ion. However the activation mechanism and physiological function of atypical PKC isozymes are less well understood. Here we show that a lipid mediator, sphingosine 1‐phosphate (S1P) controls the cellular activity of atypical PKC isozymes. First, we generated a genetically encoded reporter with the same modular structure as the original C kinase activity reporter (CKAR) but with a unique substrate sequence that allows specific visualization of atypical PKC activity in cells. Using the atypical PKC‐specific CKAR (aCKAR) we found that intracellular S1P induces the activation of atypical PKC in an S1P receptor‐independent manner. Biochemical studies reveal that S1P directly binds to the kinase domain of atypical PKC isozymes, relieving autoinhibitory constraints to activate the enzyme. In silico docking studies were used to identify potential binding sites for aPKC, one of which was validated biochemically. Our results are consistent with a model in which S1P binds the kinase domain of atypical PKC isozymes, an event that releases autoinhibitory constraints to lock the PKC in an active conformation. Our results provide a new molecular mechanism for controlling the cellular activity of atypical PKC. Support or Funding Information This study was supported by NIH to A.C.N., NIH, HHMI, NBCR, NSF to J.A.M., JSPS KAKENHI, Kobe University Grant for Japan‐US Collaboration, Nakatani Foundation Grant for Technology Development Research to T.K., JSPS KAKENHI to S.N. A.D.C. was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .